Zhang Chuanjie, Shen Yan, Gao Lili, Wang Xiaojing, Huang Da, Xie Xin, Xu Danfeng, He Hongchao
Department of Urology, Shanghai Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Research Center for Experimental Medicine, Shanghai Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Front Cell Dev Biol. 2021 Feb 11;9:622344. doi: 10.3389/fcell.2021.622344. eCollection 2021.
The aim of this study is to investigate the biological functions and the underlying mechanisms of DNA polymerase epsilon subunit 2 (POLE2) in renal cell carcinoma (RCC).
The datasets of POLE2 expression in The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) and International Cancer Genome Consortium (ICGC) databases was selected and the correlation between POLE2 and various clinicopathological parameters was analyzed. The POLE2 expression in RCC tissues was examined by immunohistochemistry. The POLE2 knockdown cell lines were constructed. and experiments were carried out to investigate the function of POLE2 on cellular biology of RCC, including cell viability assay, clone formation assay, flow cytometry, wound-healing assay, Transwell assay, qRT-PCR, Western blot, etc. Besides, microarray, co-immunoprecipitation, rescue experiment, and Western blot were used to investigate the molecular mechanisms underlying the functions of POLE2.
POLE2 was overexpressed in RCC tissues, and high expression of POLE2 was correlated with poor prognosis of RCC. Furthermore, knockdown of POLE2 significantly inhibited cell proliferation, migration, and facilitated apoptosis . experiments revealed that POLE2 attenuated RCC tumorigenesis and tumor growth. we also illuminated that stanniocalcin 1 (STC1) was a downstream gene of POLE2, which promoted the occurrence and development of RCC. Besides, knockdown of POLE2 significantly upregulated the expression levels of Bad and p21 while the expression levels of HSP70, IGF-I, IGF-II, survivin, and sTNF-R1 were significantly downregulated. Western blot analysis also showed that knockdown of POLE2 inhibited the expression levels of Cancer-related pathway proteins including p-Akt, CCND1, MAPK9, and PIK3CA.
Knockdown of POLE2 attenuates RCC cells proliferation and migration by regulating STC1, suggesting that POLE2-STC1 may become a potential target for RCC therapy.
本研究旨在探讨DNA聚合酶ε亚基2（POLE2）在肾细胞癌（RCC）中的生物学功能及潜在机制。
选取癌症基因组图谱肾透明细胞癌（TCGA-KIRC）和国际癌症基因组联盟（ICGC）数据库中POLE2表达的数据集，分析POLE2与各种临床病理参数之间的相关性。通过免疫组织化学检测RCC组织中POLE2的表达。构建POLE2敲低细胞系，并进行实验以研究POLE2对RCC细胞生物学功能的影响，包括细胞活力测定、克隆形成测定、流式细胞术、伤口愈合测定、Transwell测定、qRT-PCR、蛋白质免疫印迹等。此外，利用基因芯片、免疫共沉淀、挽救实验和蛋白质免疫印迹来研究POLE2功能的分子机制。
POLE2在RCC组织中高表达，且POLE2高表达与RCC预后不良相关。此外，敲低POLE2可显著抑制细胞增殖、迁移并促进细胞凋亡。实验表明POLE2可减弱RCC的肿瘤发生和肿瘤生长。我们还发现，鲟鱼钙蛋白1（STC1）是POLE2的下游基因，其促进了RCC的发生和发展。此外，敲低POLE2可显著上调Bad和p21的表达水平，而热休克蛋白70（HSP70）、胰岛素样生长因子-I（IGF-I）、胰岛素样生长因子-II（IGF-II）、生存素和可溶性肿瘤坏死因子受体1（sTNF-R1）的表达水平则显著下调。蛋白质免疫印迹分析还显示，敲低POLE2可抑制包括磷酸化蛋白激酶B（p-Akt）、细胞周期蛋白D1（CCND1）、丝裂原活化蛋白激酶9（MAPK9）和磷脂酰肌醇-3激酶催化亚基α（PIK3CA）在内的癌症相关信号通路蛋白的表达水平。
敲低POLE2通过调节STC1减弱RCC细胞的增殖和迁移，提示POLE2-STC1可能成为RCC治疗的潜在靶点。
